Ion exchange chromatography (IEC) is a widely used analytical technique for the chemical analysis and separation of charged molecules. IEC involves the separation of one or more analyte species from other matrix component present in a sample. The analytes are typically ionic so that they can have an ionic interaction with a stationary phase. In IEC, the stationary phase is derivatized with ionic moieties that ideally will bind to the analytes and matrix components with varying levels of affinity. An eluent is percolated through the stationary phase and competes with the analyte and matrix components for binding to the ionic moieties. The eluent is a term used to describe a liquid solution or buffer solution that is pumped into a chromatography column inlet. During this competition, the analyte and matrix components will elute off of the stationary phase as a function of time and then be subsequently detected at a detector. Examples of some typical detectors are a conductivity detector, a UV-VIS spectrophotometer, and a mass spectrometer. Over the years, IEC has developed into a powerful analytical tool that is useful for creating a healthier, cleaner, and safer environment where complex sample mixtures can be separated and analyzed for various industries such as water quality, environmental monitoring, food analysis, pharmaceutical, and biotechnology.
In the biotechnology industry, there have been numerous breakthrough discoveries in developing therapeutic drugs that are based on proteins. Monoclonal antibodies (MAbs) represent a particular type of protein therapeutic that has been successful in treating diseases such as Crohn's disease, rheumatoid arthritis, non-Hodgkin lymphoma, and metastatic breast cancer. Given the past successes in MAb technology, there is a continuing interest in developing new therapeutic applications of MAb technology, and more particularly, in accelerating the discovery, development, and screening process, which has heretofore been expensive and time consuming.
Recombinant MAbs are highly heterogeneous due to modifications such as C-terminal lysine truncation, N-terminal pyroglutamate formation, deamidation, sialylation, glycation, and glycosylation. Some of these modifications can cause a variation in the charge of a MAb. For example, deamidation and sialylation will introduce negatively charged acidic moieties on the MAb. An array of positively charged MAb variants can be created through a C-terminal lysine truncation. Traditionally, an eluent salt gradient with cation ion-exchange chromatography has been used to characterize MAb charge variants. However, method development is often required to configure the salt gradient and separation parameters every time a new MAb candidate requires characterization. For example, such parameters may include buffer salt type, buffer salt concentration, non-buffer salt type, non-buffer salt concentration, flow rates, time profile for establishing the change in proportions of the eluent solution components, and rate of pH change per unit time. Applicants believe that there is a need to develop a buffer kit and system that can provide a linear pH gradient over a wide pH range and can be used to characterize a wide variety of proteins (e.g., MAbs) with little to no modification of the separation parameters for each new candidate.